Dimeric proteins for immunization

ABSTRACT

A dimeric protein comprising a first fusion protein and a second fusion protein, wherein the first fusion protein comprises a targeting domain, a leucine zipper domain, and an antigen; and wherein the second fusion protein comprises a targeting domain, a leucine zipper domain, and optionally an antigen. Nucleic acid vectors encoding proteins of the invention are provided, particularly for use in nucleic acid vaccination.

RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.10/479,649, filed on Dec. 1, 2003, now U.S. Pat. No. 7,541,180 which isa §371 National Phase filing of PCT/IB02/03105, filed May 30, 2002 andpublished in English, which claims the benefit of GB 0113798.3, filedJun. 6, 2001, from which applications priority is claimed pursuant tothe provisions of 35 U.S.C. §§119/120. The above applications areincorporated by reference herein in their entities.

All documents cited herein are incorporated by reference in theirentirety

TECHNICAL FIELD

This invention is in the field of vaccination, and in particularantigens and vectors useful for nucleic acid vaccination. The vectorscan be used to stimulate a specific immune response against one or moreantigens.

BACKGROUND ART

Immunization with nucleic acids has the potential to circumvent many ofthe problems associated with protein vaccines.

Nucleic acid vaccines are relatively inexpensive to produce, overcomingthe high costs of production and difficulties in purification associatedwith the preparation of soluble protein antigens. Furthermore,immunization with protein antigen is associated with problems ofincorrect folding and poor induction of cell-mediated immunity. Incontrast, nucleic acid immunization generates correctly foldedconformational determinants and maximizes the presentation of processedantigenic determinants on MHC molecules, eliciting both humoral andcell-mediated immunity. Nucleic acid vaccination therefore holds greatpromise for infectious disease prophylactics and immunotherapy.

The efficacy of DNA vaccination has been successfully demonstrated inanimal models for a wide range of pathogens (Pardoll, 1995; Donnelly,1997). However, there are still major practical and theoreticalobstacles to overcome before the potential of these vaccines can berealized. Most animal model studies were carried out in rodents and itsubsequently became clear that in larger animals, in particular primatesand humans, DNA vaccination has limited efficacy (Fuller, 1997; Wang,1998). Although DNA vaccines typically elicit good cell-mediatedimmunity, they elicit poor humoral immunity, leading some to concludethat nucleic acid vaccines will not replace conventional proteinvaccines.

Several strategies to improve the immunogenicity of DNA vaccines andmanipulate the immune response have been developed, such as the use ofvarious viral, eukaryotic and combined promoters (Donnelly, 1997;Barnhart, 1998; Armand, 2000; Kwissa, 2000), the improvement oftransgene expression (Hartikka, 1996; Yew, 1997), the addition ofimmunostimulatory sequences into the vector backbone which act in vivoas a T helper 1 enhancing adjuvant (Sato, 1996), or the co-expression ofcostimulatory molecules, such as cytokines or chemokines (Leitner,2000).

However, none of these strategies has been particularly successful andthere remains a need to increase the immunogenicity of nucleic acidvaccines and enhance humoral responses elicited by nucleic acidvaccines. An object of the invention is to provide nucleic acid vaccineswhich elicit good humoral responses, making them a viable alternative toconventional vaccines.

An approach suggested by Boyle et al involves targeting antigen to sitesof immune induction by vaccination with DNA encoding the antigen as aCTLA-4 fusion protein (Boyle, 1998). CTLA-4 is expressed on activated Tcells and binds to the surface receptor B7-1 and B7-2 ofantigen-presenting cells (APCs), which are potent initiators of immuneresponses (Linsley, 1990). Deliyannis et al demonstrated that thisapproach enhanced the speed and magnitude of the immune response againsta viral antigen in mice. Vaccinated mice were found to havesignificantly reduced viral titres when faced with viral challenge(Deliyannis et al, 2000). Chaplin et al found that targeting improvedthe efficacy of a DNA vaccine against Corynebacterium in sheep (Chaplinet al, 1999).

These workers used vaccination vectors comprising a plasmid encoding anantigen fused to a CTLA-4-human immunoglobulin structure. High aviditybinding of CTLA-4 to its receptor requires CTLA-4 dimerization (Greene,1996), and this dimerization is also critical for the observedenhancement of the immune response (Lew, 2000). As a dimerizationmoiety, the authors used either the hinge and heavy chain constantdomains of human IgG1, or only the hinge regions of human IgG3.

However, this approach also results in antigen dimerization which canhave negative effects on the quality of the immune response. Forexample, monomers but not dimers of recombinant HCV glycoprotein E2,expressed in chinese hamster ovary cells, have the biological activityrequired to elicit a good antibody response (Heile, 2000). RecombinantE2 protein expressed in mammalian cells tends to form aggregates, whichare stabilised by disulphide bonds. It is likely that such aggregateswill also form when E2 is forced to be in close vicinity with a copy ofitself in the context of a dimerized fusion protein. Thus, an object ofthe invention is the provision of more sophisticated nucleic acidvectors which allow the antigen to be targeted to the preferred cells ina monomeric state.

Similarly, there is a need for nucleic acid vectors that would allow thehomodimerization or heterodimerization of the targeting moiety whilstallowing antigen heterodimerization. Taking HCV as an example again, E2heterodimerizes with E1 in vitro. When vaccinated with recombinant E1-E2heterodimers, chimpanzees develop high titres of anti-E2 antibodies inserum and are completely protected from subsequent challenge with thehomologous virus (Choo, 1994; Rosa, 1996). An object of the invention isto circumvent the inherent difficulties associated with producing E1-E2heterodimers by providing a nucleic acid vaccine encoding an E1-E2heterodimer such that the heterodimer is targeted to APCs. A furtherobject of the invention is to provide nucleic acid vaccines whichstimulate effective cell-mediated and humoral immune responses againstmore than one antigen

Monoclonal antibodies are invaluable research tools for varioustechniques such as ELISA, immunoblots, immunohistology andcytofluorimetry and are often the key to the characterization of proteinstructure, function and purification. They permit efficient purificationby affinity chromatography and allow functional characterization byblocking binding or active sites. Traditionally, the production ofspecific polyclonal and monoclonal antibodies has required thepurification and injection of significant amounts of protein fromtissues or fluids for immunization, or the injection of mammalian cellclones expressing certain markers, followed by extensive screeningprocedures. It is now possible to obtain information about a proteinfrom its cloned sequence without ever having purified it, andrecombinant forms of the protein can be expressed and purified fromcells or supernatent in various systems. However, this remains adifficult undertaking and the process of obtaining recombinant proteinin native conformation in sufficient yield and purity for immunizationand screening still remains a limiting step in the path towardsobtaining antibodies. Immunizing mice or other small animals withnucleic acid encoding the protein has been suggested as a way ofovercoming these problems (Ulvieri, 1996). However, current nucleic acidimmunization vectors do not provide sufficient yields to make thismethod viable. An object of the invention is the provision of nucleicacid vectors which allow increased antibody yields to be obtained.

DISCLOSURE OF THE INVENTION

According to a first aspect of the invention, there is provided adimeric protein comprising a first fusion protein and a second fusionprotein,

-   -   wherein the first fusion protein comprises        -   (i) a targeting domain,        -   (ii) a leucine zipper domain, and        -   (iii) an antigen;    -   and wherein the second fusion protein comprises        -   (i) a targeting domain        -   (ii) a leucine zipper domain, and        -   (iii) optionally an antigen.

The targeting domain in the first and the second fusion proteins may befused to either the C-terminal or the N-terminal of the leucine zipperdomain with the antigen domain fused to the other terminal of theleucine zipper domain. The order of fusion in the first and the secondfusion proteins is the same. Hence if the targeting domain of the firstfusion protein is fused to the C-terminal of the leucine zipper domainwith the antigen fused to the N-terminal of the leucine zipper domain,the targeting domain of the second fusion protein is also fused to theC-terminal of the leucine zipper domain with an antigen optionally fusedto the N-terminal of the leucine zipper domain. Similarly, if thetargeting domain of the first fusion protein is fused to the N-terminalof the leucine zipper domain with the antigen fused to the C-terminal ofthe leucine zipper domain, the targeting domain of the second fusionprotein is also fused to the N-terminal of the leucine zipper domainwith an antigen optionally fused to the C-terminal of the leucine zipperdomain.

The dimeric protein may be targeted to antigen presenting cells (APCs),such as MHC II+ B-cells, dendritic cells and monocyte macrophages. Thedimeric protein can bind to proteins on the surface of the APCs via thetargeting proteins. Binding of the dimeric protein to APCs targets theantigen(s) in the dimeric protein to those cells, stimulating anenhanced immune response to the antigen(s).

The targeting domains in the first and second fusion proteins may be thesame or different. Where the targeting domains are the same, preferablythey will be CTLA4 or a fragment thereof, most preferably the V-likedomain. As an alternative, the targeting domain can be an antibody whichwill bind to a protein on the surface of the APC. Recently, a reporterantigen linked to a monoclonal antibody that binds to the CD11c moleculeon the surface of murine dendritic cells was used for immunisation. Thestudy demonstrated that a single-step delivery of small amounts ofprotein antigen targeted to dendritic cells in vivo can give very rapidand high antibody responses and strongly supports the general concept oftargeting antigens to receptors. In such a situation, the targetingprotein in one fusion protein can be the light chain of the antibody andthe targeting protein in the other fusion protein can be the heavy chainof the antibody.

The term “leucine zipper domain” is used to denote a commonly recogniseddimerisation domain characterised by the presence of a leucine residueevery seventh residue in a stretch of approximately 35 residues. Leucinezipper domains form dimers held together by an alpha-helical coiledcoil. A coiled coil has 3.5 residues per turn, which means that everyseventh residue occupies an equivalent position with respect to thehelix axis. The regular array of leucines inside the coiled coilstabilizes the structure by hydrophobic and Van der Waals interactions.

Leucine zipper domains in first fusion protein and the second fusionprotein may be the same or different. The leucine zipper domains may beisolated from natural proteins known to contain such domains, such astranscription factors. Preferably, where both leucine zipper domains arethe same, they are from the transcription factor GCN4. Alternatively,where the leucine zipper domains are different, one leucine zipperdomain comes from the transcription factor fos and the second from thetranscription factor jun. The leucine zipper domains may also bedesigned and synthesised artificially, using standard techniques forsynthesis and design known in the art.

The antigens of the first and second fusion proteins may be the same ordifferent. If they are the same, the antigen will be targeted to APCs inhomodimeric form or, if it does not form a dimer, in dual monomericform. If they are different, the two antigens can be targeted to APCs inheterodimeric form or, if they do not form a dimer, in separatemonomeric form. Heterodimeric antigens may be used advantageously topromote an immune response against antigens which is correctly foldedonly in the context of a heterodimer. Alternatively, an immune responseagainst both antigens may be stimulated. Finally, only one of the fusionproteins need include an antigen, such that the antigen is targeted toAPCs as a single monomer. The dimeric proteins of the inventiontherefore enable the antigen(s) to be targeted to APCs in singlemonomeric, dual monomeric, homodimeric, heterodimeric or separatemonomeric form.

The antigen or antigens may be any protein. Preferably, the antigens areselected from tumour antigens, bacterial antigens, viral antigens orparasitic antigens. Examples of antigens which may be employed includeA/B toxins such as cholera toxin or E coli heat labile toxin, HepatitisC antigens (e.g. hepatitis B surface antigen), pertussis toxin, viralsplice proteins and Hepatitis C antigens. Preferably, viral antigens areHCV antigens. Most preferably, the antigen in the first fusion proteinis the HCV E1 protein and the antigen in the second fusion protein isthe HCV E2 protein.

The fusion proteins may further comprise linker sequences, such asglycine linkers, between the targeting protein and the leucine zipperdomain and/or the leucine zipper domain and the antigen.

According to a further aspect of the invention there is provided amonomeric protein comprising:

-   -   (i) a targeting domain    -   (ii) a leucine zipper domain and    -   (iii) an antigen.

Two such proteins can dimerise via their leucine zipper domains to forma dimeric protein. The monomeric proteins which dimerise to form adimeric protein may be the same or they may be different. The dimericproteins formed by the dimerisation of two such monomeric proteinsinclude the dimeric proteins described above.

According to a further aspect of the invention, there is provided anucleic acid vector encoding a protein of the invention. The vector mayencode one or more fusion proteins. Where the vector encodes one fusionprotein, this can be itself used for the expression of homodimers, orcan be used in combination with a different second vector for theexpression of heterodimers. Where the vector encodes two fusionproteins, dimers can be expressed using a single vector.

The invention also provides a kit comprising two or more vectors of theinvention.

Where the nucleic acid vector is a DNA vector, the vector will comprisea promoter. A “promoter” is a region of DNA that signals RNA polymerasebinding and the initiation of transcription. The promoters used in thevectors of the invention must be functional in mammalian cells. That is,they must be able to direct efficient transcription of the fusionproteins encoded by the vector. Preferably, the promoters employed inthe vectors of the invention are selected from the group consisting of aCMV promoter or an alphavirus promoter In the case of a kit comprisingtwo monomeric vectors, the promoters for the two monomeric proteinsencoded by the two vectors may be the same or different.

Where the vector encodes two fusion proteins, these may be translatedfrom separate transcripts, or the transcript may comprise an internalribosome entry site (IRES; Ramos, 1999) to enable translation of the twofusion proteins from a single transcript.

Optionally, the nucleic vector(s) of the invention may further comprisesequences encoding proteins that promote secretion of the monomeric ordimeric proteins they encode or target them to specific intracellularcompartments. For example, the human tissue-type plasminogen activator(TPA) signal/pro sequence (Pennica, 1983) can be fused to the N-terminusof the targeting protein, such as CTLA-4. This sequence has been shownto be very efficient in facilitating protein transport not only into theER, but from the ER to the Golgi apparatus. It enables even proteinswhich are blocked in their secretion pathway to bypass the ER retentionmechanism. Alternative sequences may be used such as leader sequences,the ER retention sequence KDEL (SEQ ID NO:22) and the GPI anchor signalwhich promotes surface expression. The targeting protein should remainactive in the presence of such additional sequences.

The nucleic acid vectors of the invention have a number of additionalcharacteristics common to nucleic acid vectors in general.

The vectors may comprise a bacterial origin of replication to providehigh copy numbers during production. An “origin of replication” is anucleic sequence that initiates and regulates replication of nucleicacids, such as an expression vector. The origin of replication behavesas an autonomous unit of nucleic acid replication within a cell, capableof replication under its own control. Preferably, this origin ofreplication is derived from the pUC19 (Yanisch-Perron, 1985) backbone ofpCMVβ and provides high copy numbers in E. coli.

The vector may further comprise a selection marker for selection in E.coli. This gene may be an antibiotic resistance gene or a gene involvedin a metabolic pathway. Preferably, said gene is the asd gene ofSalmonella typhimurium, which encodes aspartate β-semialdehydedehydrogenase, an enzyme common to the biosynthetic pathways of lysine,threonine and methionine, as well as diaminopimelic acid, an essentialconstituent of the peptidoglycan cell wall of gram negative bacteria(Scleifer, 1972).

The nucleic acid vectors of the invention may also comprise a multiplecloning site to facilitate the cloning of antigens. The multiple cloningsite contains a number of unique endonuclease restriction sites and isinserted in both directions.

The vectors may further comprise sequences to permit splicing and 3′polyadenylation of the transcript. Preferably, a fragment containing twointrons used by SV40 to splice the viral 16S and 19S late mRNAs isplaced upstream of the cloning site and a 196 bp fragment containing alate region polyA signal occurs downstream of the cloning site (Okayama,1983).

The nucleic acid vectors may further comprise CG motifs which have anadjuvant effect, such as those described in WO 00/50075.

The functional prokaryotic and eukaryotic units (ori, selection marker,promoter, splice site and polyA signal) are preferably flanked by uniquerestriction sites, allowing their rapid removal and replacement or theintroduction of additional elements for research or efficiency purposes,such as immunoregulatory sequences or genes.

A further aspect of the invention is a host cell comprising a vector orvectors of the invention as described above.

The vectors and compositions of the invention are primarily for use innucleic acid vaccination. A further aspect of the invention is thereforea pharmaceutical composition comprising a therapeutically effectiveamount of the nucleic acid vector or vectors of the invention orcompositions comprising said vectors in combination withpharmaceutically acceptable carriers.

The term “nucleic acid” includes DNA and RNA, and also their analogues,such as those containing modified backbones, and also peptide nucleicacids (PNA) etc.

By “therapeutically effective amount”, it is meant that theadministration of that amount to an individual, either in a single doseor as part of a series, is effective for treatment or prevention. Thisamount varies depending upon the health and physical condition of theindividual to be treated, the taxonomic group of individual to betreated (e.g. nonhuman primate, primate, etc.), the capacity of theindividual's immune system to synthesize antibodies, the degree ofprotection desired, the formulation of the vaccine, the treatingdoctor's assessment of the medical situation, and other relevantfactors. It is expected that the amount will fall in a relatively broadrange that can be determined through routine trials.

“Pharmaceutically acceptable carriers” include any carrier that does notitself induce the production of antibodies harmful to the individualreceiving the composition. Suitable carriers are typically large, slowlymetabolized macromolecules such as proteins, polysaccharides, polylacticacids, polyglycolic acids, polymeric amino acids, amino acid copolymers,lipid aggregates (such as oil droplets or liposomes), and inactive virusparticles. Such carriers are well known to those of ordinary skill inthe art. Additionally, these carriers may function as immunostimulatingagents (“adjuvants”).

Preferred adjuvants to enhance effectiveness of the composition include,but are not limited to: (1) aluminum salts (alum), such as aluminumhydroxide, aluminum phosphate, aluminum sulfate, etc; (2) oil-in-wateremulsion formulations (with or without other specific immunostimulatingagents such as muramyl peptides (see below) or bacterial cell wallcomponents), such as for example (a) MF59™ (WO 90/14837; Powell &Newman, 1995), containing 5% Squalene, 0.5% TWEEN 80 polyoxyethylenesorbitan monooleate surfactant, and 0.5% SPAN 85 sorbitan trioleateformulated into submicron particles using a microfluidizer such as Model110Y microfluidizer (Microfluidics, Newton, Mass.), (b) SAF, containing10% Squalane, 0.4% TWEEN 80 surfactant, 5% PLURONIC L121 block polymersurfactant, and thr-MDP (see below) either microfluidized into asubmicron emulsion or vortexed to generate a larger particle sizeemulsion, and (c) RIBI adjuvant system (RAS), (Ribi Immunochem,Hamilton, Mont.) containing 2% Squalene, 0.2% TWEEN 80 surfactant, andone or more bacterial cell wall components from the group consisting ofmonophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wallskeleton (CWS), preferably MPL+CWS (Detox™); (3) saponin adjuvants, suchas Stimulon™ (Cambridge Bioscience, Worcester, Mass.) may be used orparticles generated therefrom such as ISCOMs (immunostimulatingcomplexes); (4) Complete Freund's Adjuvant (CFA) and Incomplete Freund'sAdjuvant (IFA); (5) cytokines, such as interleukins (e.g. IL-1, IL-2,IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g. gammainterferon), macrophage colony stimulating factor (M-CSF), tumornecrosis factor (TNF), etc; and (6) other substances that act asimmunostimulating agents to enhance the effectiveness of thecomposition. Alum and MF59™ are preferred.

As mentioned above, muramyl peptides include, but are not limited to,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(MTP-PE), etc.

The pharmaceutical compositions (e.g. the polynucleotide vector(s),pharmaceutically acceptable carrier, and adjuvant) typically willcontain diluents, such as water, saline, glycerol, ethanol, etc.Additionally, auxiliary substances, such as wetting or emulsifyingagents, pH buffering substances, and the like, may be present in suchvehicles.

A further aspect of the invention is a method of stimulating an immuneresponse to a specific antigen or antigens by administering thevector(s), compositions or pharmaceutical compositions of the inventionto a subject. The invention further provides the use of thepharmaceutical compositions, compositions and vector(s) of the inventionin the manufacture of a medicament to stimulate an immune responseagainst a specific antigen or specific antigens encoded by the vector orvectors.

Typically, the pharmaceutical compositions for use as vaccines areprepared as injectables, either as liquid solutions or suspensions;solid forms suitable for solution in, or suspension in, liquid vehiclesprior to injection may also be prepared. The preparation also may beemulsified or encapsulated in liposomes for enhanced adjuvant effect.

Liposomes that can act as nucleic acid delivery vehicles are describedin U.S. Pat. No. 5,422,120, WO95/13796, WO94/23697, WO91/14445 andEP-524,968. As described in U.S. Ser. No. 60/023,867, nucleic acidsequences encoding a polypeptide can be inserted into conventionalvectors that contain conventional control sequences for high levelexpression, and then be incubated with synthetic polynucleotide transfermolecules such as polymeric DNA-binding cations like polylysine,protamine, and albumin, linked to cell targeting ligands such asasialoorosomucoid, insulin, galactose, lactose, or transferrin. Otherdelivery systems include the use of liposomes to encapsulate DNAcomprising the nucleotide encoding the fusion protein under the controlof a variety of tissue-specific or ubiquitously-active promoters.

The nucleic acid vectors of the invention can be adsorbed onto thesurface of microparticles, in particular PLG microparticles, asdescribed in WO98/33487.

The pharmaceutical compositions are conventionally administeredparenterally, e.g. by injection, either subcutaneously, intramuscularly,or transdermally/transcutaneously (e.g. WO98/20734). Additionalformulations suitable for other modes of administration include oral andpulmonary formulations, suppositories, and transdermal applications.Dosage treatment may be a single dose schedule or a multiple doseschedule. The vaccine may be administered in conjunction with otherimmunoregulatory agents.

The vectors of the invention may also be administered as naked DNA.Exemplary naked DNA introduction methods are described in WO90/11092 andU.S. Pat. No. 5,580,859. Uptake efficiency may be improved usingbiodegradable latex beads. DNA coated latex beads are efficientlytransported into cells after endocytosis initiation by the beads. Themethod may be improved further by treatment of the beads to increasehydrophobicity and thereby facilitate disruption of the endosome andrelease of the DNA into the cytoplasm.

Other conventional methods for polynucleotide delivery that can be usedfor delivery of the vector(s) include, for example, use of hand-heldparticle gun, as described in U.S. Pat. No. 5,149,655; or use ofionizing radiation for activating transferred polynucleotide, asdescribed in U.S. Pat. No. 5,206,152 and WO92/11033

Exemplary liposome and polycationic delivery vehicles are thosedescribed in U.S. Pat. Nos. 5,422,120 and 4,762,915; in WO 95/13796;WO94/23697; and WO91/14445; in EP-0524968; and in Stryer, 1975; Szoka,1980; Bayer, 1979; Rivnay, 1987; Wang, 1987; and Plant, 1989.

Once formulated, the polynucleotide compositions of the invention can be(1) administered directly to the subject as described above; or (2)delivered in vitro.

The vectors and pharmaceutical compositions of the present inventionhave a use in vitro for producing increased yields of antibodies. Asmentioned in the introduction, monoclonal and polyclonal antibodies arewidely used in research and diagnostics. However, the production of suchantibodies is often time consuming and depends on the purification ofthe protein in its native form. The vectors and pharmaceuticalcompositions of the current invention allow the production of antibodiesto an antigen when the only information known about that antigen is itsgene sequence. A further aspect of the invention is a method ofstimulating an immune response to a specific antigen in vitro byadministration of the vectors, compositions and pharmaceuticalcompositions of the invention. Antibodies obtained by the use of thismethod comprise a further aspect of the invention.

Generally, delivery of nucleic acids for in vitro applications can beaccomplished by the following procedures, for example, dextran-mediatedtransfection, calcium phosphate precipitation, polybrene mediatedtransfection, protoplast fusion, electroporation, encapsulation of thepolynucleotide(s) in liposomes, and direct microinjection of the DNAinto nuclei, all well known in the art.

In addition to the pharmaceutically acceptable carriers and saltsdescribed above, the following additional agents can be used withpharmaceutical compositions.

A. Polypeptides

One example are polypeptides which include, without limitation:asioloorosomucoid (ASOR); transferrin; asialoglycoproteins; antibodies;antibody fragments; ferritin; interleukins; interferons, granulocyte,macrophage colony stimulating factor (GM-CSF), granulocyte colonystimulating factor (G-CSF), macrophage colony stimulating factor(M-CSF), stem cell factor and erythropoietin. Viral antigens, such asenvelope proteins, can also be used. Also, proteins from other invasiveorganisms, such as the 17 amino acid peptide from the circumsporozoiteprotein of plasmodium falciparum known as RII.

B. Hormones, Vitamins, Etc.

Other groups that can be included are, for example: hormones, steroids,androgens, estrogens, thyroid hormone, or vitamins, folic acid.

C. Polyalkylenes, Polysaccharides, Etc.

Also, polyalkylene glycol can be included with the desiredpolynucleotides/polypeptides. In a preferred embodiment, thepolyalkylene glycol is polyethlylene glycol. In addition, mono-, di-, orpolysaccharides can be included. In a preferred embodiment of thisaspect, the polysaccharide is dextran or DEAE-dextran. Also, chitosanand poly(lactide-co-glycolide)

D. Lipids, and Liposomes

The desired polynucleotide can also be encapsulated in lipids orpackaged in liposomes prior to delivery to the subject or to cellsderived therefrom.

Lipid encapsulation is generally accomplished using liposomes which areable to stably bind or entrap and retain nucleic acid. The ratio ofcondensed polynucleotide to lipid preparation can vary but willgenerally be around 1:1 (mg DNA:micromoles lipid), or more of lipid. Fora review of the use of liposomes as carriers for delivery of nucleicacids, see, Hug and Sleight, 1991; and Straubinger, 1983.

Liposomal preparations for use in the present invention include cationic(positively charged), anionic (negatively charged) and neutralpreparations. Cationic liposomes have been shown to mediateintracellular delivery of plasmid DNA (Feigner, 1987); mRNA (Malone,1989); and purified transcription factors (Debs, 1990), in functionalform.

Cationic liposomes are readily available e.g.N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes areavailable under the trademark Lipofectin, from GIBCO BRL, Grand Island,N.Y. (See, also, Feigner supra). Other commercially available liposomesinclude transfectace (DDAB/DOPE) and DOTAP/DOPE (Boehringer). Othercationic liposomes can be prepared from readily available materialsusing techniques well known in the art. See, e.g. Szoka, 1978;WO90/11092 for a description of the synthesis of DOTAP(1,2-bis(oleoyloxy)-3-(trimethylammonio) propane) liposomes.

Similarly, anionic and neutral liposomes are readily available, such asfrom Avanti Polar Lipids (Birmingham, Ala.), or can be easily preparedusing readily available materials. Such materials include phosphatidylcholine, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidyl glycerol (DOPG),dioleoylphoshatidyl ethanolamine (DOPE), among others. These materialscan also be mixed with the DOTMA and DOTAP starting materials inappropriate ratios. Methods for making liposomes using these materialsare well known in the art.

The liposomes can comprise multilammelar vesicles (MLVs), smallunilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs). Thevarious liposome-nucleic acid complexes are prepared using methods knownin the art. See e.g. Straubinger, 1983; Szoka, 1978; Papahadjopoulos,1975; Wilson, 1979; Deamer & Bangham, 1976; Ostro, 1977; Fraley, 1979;Enoch & Strittmatter, 1979; Fraley, 1980; Szoka & Papahadjopoulos, 1978;and Schaefer-Ridder, 1982.

E. Lipoproteins

In addition, lipoproteins can be included with the polynucleotide to bedelivered. Examples of lipoproteins to be utilized include:chylomicrons, HDL, IDL, LDL, and VLDL. Mutants, fragments, or fusions ofthese proteins can also be used. Also, modifications of naturallyoccurring lipoproteins can be used, such as acetylated LDL. Theselipoproteins can target the delivery of polynucleotides to cellsexpressing lipoprotein receptors. Preferably, if lipoproteins areincluding with the polynucleotide to be delivered, no other targetingligand is included in the composition.

Naturally occurring lipoproteins comprise a lipid and a protein portion.The protein portion are known as apoproteins. At the present,apoproteins A, B, C, D, and E have been isolated and identified. Atleast two of these contain several proteins, designated by Romannumerals, AI, AII, AIV; CI, CII, CIII.

A lipoprotein can comprise more than one apoprotein. For example,naturally occurring chylomicrons comprises of A, B, C, and E, over timethese lipoproteins lose A and acquire C and E apoproteins. VLDLcomprises A, B, C, and E apoproteins, LDL comprises apoprotein B; andHDL comprises apoproteins A, C, and E.

The amino acid of these apoproteins are known and are described in, forexample, Breslow, 1985; Law, 1986; Chen, 1986; Kane, 1980; and Utermann,1984.

Lipoproteins contain a variety of lipids including, triglycerides,cholesterol (free and esters), and phospholipids. The composition of thelipids varies in naturally occurring lipoproteins. For example,chylomicrons comprise mainly triglycerides. A more detailed descriptionof the lipid content of naturally occurring lipoproteins can be found,for example, in Meth. Enzymol. 128 (1986). The composition of the lipidsare chosen to aid in conformation of the apoprotein for receptor bindingactivity. The composition of lipids can also be chosen to facilitatehydrophobic interaction and association with the polynucleotide bindingmolecule.

Naturally occurring lipoproteins can be isolated from serum byultracentrifugation, for instance. Such methods are described in Pitas,1980 and Mahey, 1979. Lipoproteins can also be produced by in vitro orrecombinant methods by expression of the apoprotein genes in a desiredhost cell. See, for example, Atkinson, 1986. Lipoproteins can also bepurchased from commercial suppliers, such as Biomedical Techniologies,Inc., Stoughton, Mass., USA. Further description of lipoproteins can befound in Zuckermann et al. PCT/US97/14465.

F. Polycationic Agents

Polycationic agents can be included, with or without lipoprotein, in acomposition with the desired polynucleotide to be delivered.

Polycationic agents, typically, exhibit a net positive charge atphysiological relevant pH and are capable of neutralizing the electricalcharge of nucleic acids to facilitate delivery to a desired location.These agents have both in vitro, ex vivo, and in vivo applications.Polycationic agents can be used to deliver nucleic acids to a livingsubject either intramuscularly, subcutaneously, etc.

The following are examples of useful polypeptides as polycationicagents: polylysine, polyarginine, polyornithine, and protamine. Otherexamples include histones, protamines, human serum albumin, DNA bindingproteins, non-histone chromosomal proteins, coat proteins from DNAviruses, such as (X174, transcriptional factors also contain domainsthat bind DNA and therefore may be useful as nucleic aid condensingagents. Briefly, transcriptional factors such as C/CEBP, c-jun, c-fos,AP-1, AP-2, AP-3, CPF, Prot-1, Sp-1, Oct-1, Oct-2, CREP, and TFIIDcontain basic domains that bind DNA sequences.

Organic polycationic agents include: spermine, spermidine, andpurtrescine.

The dimensions and of the physical properties of a polycationic agentcan be extrapolated from the list above, to construct other polypeptidepolycationic agents or to produce synthetic polycationic agents.

Synthetic polycationic agents which are useful include, for example,DEAE-dextran, polybrene. Lipofectin™, and lipofectAMINE™ are monomersthat form polycationic complexes when combined withpolynucleotides/polypeptides.

In general, the practice of the invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,recombinant DNA, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature e.g. Sambrook,1989; Glover, 1985; Gait, 1984; Hames & Higgins, 1984(a); Hames &Higgins 1984(b); Freshney, 1986; IRL Press, 1986; Perbal, 1984; theMethods in Enzymology series (Academic Press, Inc.), especially volumes154 & 155; Miller and Calos, 1987; Mayer and Walker, 1987; Scopes, 1987,Weir and Blackwell, 1986.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: The DNA vaccination vector pAC-FN/NF (starting point vector)

FIG. 2: The DNA vaccination vector pAC-AH (final vector) encoding theamino acid sequence of SEQ ID NO:21.

Various aspects and embodiments of the present invention will now bedescribed in some detail. It will be appreciated that modification ofdetail may be made without departing from the spirit and scope of theinvention.

EXAMPLES

I) Construction of Pac-FN-NF

The mammalian reporter vector pCMVβ (Clontech, Palo Alto, Calif.) wasdigested with Not I to remove the β-galactosidase reporter gene. Thelinearized vector was purified from a 1% agarose gel using the Qiaex IIgel extraction kit (Qiagen, Hilden, Germany) and religated with T4 DNAligase, resulting in the vector pCMV-N. To introduce multiple cloningsites (MCS), oligonucleotides were generated using an ABI 394 DNAsynthesizer (Perkin Elmer), using reagents from Cruachem (Glasgow), andpurified following the manufacturer's instructions. Two oligomers of 71nucleotides length (mcs1 and mcs2) (table 1) were annealed at equimolarconditions in T4 ligase buffer by heating to 70° C. for 5 minutes,slowly cooling down to 30° C., and a final chill on ice. The resultingdouble stranded MCS (MCS-FN/NF)) with 5′-GGCC-overhangs was insertedinto the Not I site of Pcmv-N. the mcs1 oligomer was labeled with[γ-³²P]-ATP by polynucleotide kinase (New England Biolabs) and used toidentify positive clones by colony hybridisation. Clones were tested forthe direction of the MCS by restriction digestions. The resultingvectors were designated pCMV-FN and pCMV-NF. The pUC19 ori was amplifiedby PCR with sense primer ori1 and antisense primer ori2 (table 1) onpCMVβ, and the PCR product was digested with SalI and HindIII. The asdgene of Salmonella typhimurium was amplified by PCR with sense primerask1 and antisense primer asd2 (see table 1) on pYA3137, which waskindly provided by Roy Curtiss III, Washington University, St. Louis,Mo. The PCXR product was digested with HindIII and PstI. All PCRamplifications were carried out using a GeneAmp PCR System 2400 machine(Perkin Elmer) and using Pwo Polymerase (Boehringer Mannheim, Monza,Italy).

Both pCMV-FN and pCMV-NF were digested with PstI and SalI and theeukaryotic expression cassettes of 1086 basepairs (bp) length werepurified from a 1% agarose gel. The digested PCR products ori (675 bp)and asd (1739 bp) were purified on MICROSPINS-400 HR columns and ligatedto the 1086 by fragments. The resulting pasmids are referred to aspAC-FN and pAC-NF (3500 bp). All unique restriction sites were tested bythe corresponding restriction endonucleases and the polylinker sequencewas confirmed using an ABI cycle sequencer.

II) Construction of pAC-AH

In a first step, a murine immunostimulatory sequence (ISS) of 30 bylength (Hartmann, 2000) was introduced into the HindIII site of pAC-FN.Annealing of the oligonucleotides iss1 and iss2 (table 2) results in aHindIII-compatible 5′-AGCT overhang. To facilitate screening, theHindIII site is eliminated upon insertion of the double strandedoligonucleotide, and a unique NheI site is introduced. The resultingplasmid of 3532 by length was designated pAC-FN-ISS, and the newselement was confirmed by sequencing.

In a second step, the V-like domain of mouse CTLA-4 was amplified byRT-PCR from splenocytes of a female C57BL/6 mouse. The cells wereresuspended by passage through a Falcon 70 μm mesh size nylon cellstrainer and further purified by a standard Ficoll gradient(lympholyte-M). 5×10⁶ cells were cultured per well of a six-well tissueculture cluster (Costar) in 3 ml RPMI-1640, 2 mM L-glutamine, 100 unitspenicillin per ml, 100μ streptomycin per ml, 10% FBS, and 100 units ofrecombinant human interleukin-2 (Chiron) per ml for 24 hours at 37° C.and 9% CO₂ atmosphere. 40×10⁶ cells were washed with PBS, and RNA wasextracted using Trizol reagent (GibcoVRL), following the manufacturer'sinstructions. First strand cDNA was generated using components of theSUPERSCRIPT CHOICE system for cDNA synthesis (GibcoBRL). 2 μg of totalRNA were annealed to 500 ng of oligo(dT)₁₂₋₁₈ primer. First strandsynthesis was performed for 50 minutes at 42° C. with 200 unitsSUPERSCRIPT II reverse transcriptase in first strand buffer (0.1 MDithiothreitol (DTT)-10 mM each dNTP). The enzyme was subsequentlyinactivated for 15 minutes at 70° C., and RNA was digested for 20minutes 37° C. with 1 μg RNAse A. The V-like domain of mouse CTLA-4 wasamplified by PCR with sense primer ctla41 and antisense primer ctla42(table 2) using one tenth of the generated first strand cDNA astemplate, generating a fragment of 375 by length.

In a third step, the mouse CTLA-4 domain was fused on the 5′-end to thetissue plasminogen activator (TPA) signal sequence and on the 3′-end viaglycine linkers to the GCN4 leucine zipper and to a new MCS. This wasachieved by overlapping PCR amplifications using the following(sense-antisense) primer pairs (table 2): fus1-fus2 (leading to a 443 byproduct), fus3-fus4 (508 bp), fus5-fus-6 (575 bp), fus7-fus8 (633 bp),fus7-fus9 663 bp), and fus7-fus10 (697 bp). After each step, the PCRproduct was purified from 2% agarose gel. The final 697 by product wascloned into the FseI and Nod sites of Pac-FN-ISS, and the resultingconstruct was designated Pac-AH. All unique restriction sites in pAC-AHwere confirmed using the corresponding restriction endonucleases, andthe new sequence between Fse and NotI was verified by sequencing.

III) Strains, Media, Growth Conditions, and DNA Preparation

pCMVβ-derived constructs were transformed by electroporation andamplified in electrocompetent Escherichia coli (E. coli) XL1-Blue MRF′(Stratagene). Untransformed bacteria were frown in LB mediumsupplemented with 12.5 μg/ml tetracycline, for the selection oftransformed bacteria 100 μg/ml ampicillin was added. pAC vectors weretransformed and amplified in E. coli K12 6212 (Φ80d lacZ ΔM15 deoRΔ(lacZYA-argF) U169 sup E44 N gyrA recA1 relA1 endA1 ΔasdA4Δ[zhf-2::Tn10] hsdR17 (r m⁺, provided by R. Curtis III). Medium used forgrowth of untransformed bacteria was LB medium supplemented withdiaminopimelic acid (DAP) at 50 μg/ml. After transformation byelectroporation, bacteria were incubated at 37° C. for 30 minutes in SOCmedium (Sambrook, 1989) supplemented with 50 μg DAP per ml, washed twicewith SOC medium, and plated on LB agar for selection. Selected cloneswere grown in LB medium and large-scale preparations of supercoiledpAC-derived vectors were carried out either by the standard alkalinelysis procedure, two purification steps with cesium chloride-ethidiumbromide gradients, n-butyl-alcohol extraction, isopropyl-alcoholprecipitation, and ialysis into TE buffer (Sambrook, 1989) or by the useof the ENDOFREE PLASMID MEGA kit for plasmid purification (Quiagen),following the suppliers protocol. DNA vaccines in TE buffer wereethyl-alcohol precipitated with sterile filtered 0.3 M sodium chloridefor storage, and resuspended in endotoxin free PBS prior toimmunization.

TABLE 1 Oligonucleotides used for the construction of pAC-FN/NF.Restriction sites in PCR primers used to assemble on and asd to theeukaryotic expression box are underlined and in italics. mcs1:5′-GGCCGCACGCGTACTAGTGGGCCCGGGCGTACGCTTAAGAATCGATATCGGTACCAGATCTGAATTCGGCC-3′ (SEQ ID NO: 1) mcs2:5′-GGCCGGCCGAATTCAGATCTGGTACCGATATCGATTCTTAAGCGTACGCCCGGGCCCACTAGTACGCGTGC-3′ (SEQ ID NO: 2) ori1: 5′-ATCGTA GTCGACGCGTTGCTGGCGTTTTTCC-3′ (SEQ ID NO: 3) ori2: 5′-ATCGTA AAGCTTATCCCTTAACGTGAGTTTTCG-3′ (SEQ ID NO: 4) asd1: 5′TACGC CTGCAGGGATCTTCCCTAAATTTAA-3′ (SEQ ID NO: 5) asd2: 5′-TACGC AAGCTTTCCAATTCAACATCAGGTA-3′ (SEQ ID NO: 6)

TABLE 2 oligonucleotides used for the construction of Pac-AH. Sequencesannealing in the first cycles of the overlapping PCR procedure are boldand underlined. Restriction sites used for screening (NheI in iss1 andiss2) and for cloning (FseI in fus7 and NotI in fus 10) are in italicsand underlined. iss1: 5′-AGCTA GCTAGC AACGTCAGGAACGTCATGGAT-3′ (SEQ IDNO: 7) iss2: 5′-AGCTATCCATGACGTTCGACGTT GCTAGCT-3′ (SEQ ID NO: 8)ctla41: 5′-GCCATACAGGTGACCCAACC-3′ (SEQ ID NO: 9) ctla42:5′-GTCAGAATCCGGGCATGG-3′ (SEQ ID NO: 10) fus1:5′-GAAATCCAATGCCCGATTCAGAAGAGCCAGATCT GCCATACAGGTGACC -3′ (SEQ ID NO:11) fus2: 5′-TGTTTCATGCGTCCACCGCCTCCTCCATCGAT GTCAGAATCCGGGC -3′ (SEQ IDNO: 12) fus3: 5′-TGTGGAGCAGTCTTCGTTTCGCCCAGCCAG GAAATCCATGCCCG -3 (SEQID NO: 13) fus4: 5′-TTTTCGAAAGAAGCTCTTCAACTTTATCTTCCAGC TGTTTCATGCGGTCC-3′ (SEQ ID NO: 14) fus5: 5′-AAGAGAGGGCTCTGCTGTGTGCTGCTGCTTGTGGAGCAGTCTTCG -3′ (SEQ ID NO: 15) fus6:5′-TTTTTGAGGCGCGCAACTTCATTTTCGAGGTGGTGGTAGT TTTTCGAAAGAAAGAAGC -3′ (SEQID NO: 16) fus7: 5′-TAAATCAT GGCCGGCC GCCATGGATGCAATG AAGAGAGGCTCTGC-3′(SEQ ID NO: 17) fus8: 5′-CCTCCACCACCGCGTTCACCAACAACAAGT TTTTTGAGGCGCGC-3′ (SEQ ID NO: 18) fus9: 5′-CTTCCAACTAGTCCTGAATTCCCGGGCCCGCTTCCAACTAGTCC -3′ (SEQ ID NO: 19) fus10: 5′-ATAGTTTA GCGGCCGCTTAACTATTCACTATAAG CTTCCAACTAGTCC -3′ (SEQ ID NO: 20)

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1. A dimeric protein comprising a first fusion protein and a secondfusion protein, wherein the first fusion protein comprises (i) atargeting domain that comprises CTLA-4 or a fragment thereof, (ii) aleucine zipper domain, and (iii) an antigen; and wherein the secondfusion protein comprises: (i) a targeting domain that comprises CTLA-4or a fragment thereof, (ii) a leucine zipper domain, and (iii)optionally an antigen.
 2. A dimeric protein according to claim 1 whichis targeted to antigen presenting cells via the targeting domains.
 3. Adimeric protein according to claim 1 or claim 2 where the targetingdomains in the first and the second fusion proteins are the same.
 4. Adimeric protein according to claim 1 or claim 2 where the targetingdomains in the first and the second fusion proteins are different.
 5. Adimeric protein according to claim 1 wherein the leucine zipper domainsin the first fusion protein and the second fusion protein are the same.6. A dimeric protein according to claim 1 wherein the leucine zipperdomains in the first fusion protein and the second fusion protein aredifferent.
 7. A dimeric protein according to claim 5 wherein bothleucine zipper domains are derived from the transcription factor GCN4.8. A dimeric protein according to claim 6 wherein one leucine zipperdomain is derived from the transcription factor fos and the other isderived from the transcription factor jun.
 9. A dimeric proteinaccording to claim 1 wherein the antigen in the first fusion protein andthe antigen in the second fusion protein are the same.
 10. A dimericprotein according to claim 1 wherein the antigen in the first fusionprotein and the antigen in the second fusion protein are different. 11.A dimeric protein according to claim 1 wherein the second fusion proteindoes not comprise an antigen.
 12. A fusion protein comprising: (i) atargeting domain that comprises CTLA-4 or a fragment thereof (ii) aleucine zipper domain and (iii) an antigen.
 13. A dimeric proteinaccording to claim 1, wherein the first fusion protein or the secondfusion protein comprises a GCN4 leucine zipper domain.
 14. A dimericprotein according to claim 1, wherein the first fusion protein or thesecond fusion protein comprises a viral antigen.
 15. A dimeric proteinaccording to claim 1, wherein the fragment comprises the CTLA-4 V-likedomain.
 16. A fusion protein according to claim 12, wherein the fusionprotein comprises a GCN4 leucine zipper domain.
 17. A fusion proteinaccording to claim 12, wherein the fusion protein comprises a viralantigen.
 18. A fusion protein according to claim 12, wherein thefragment comprises the CTLA-4 V-like domain.